CN102089662B - High capacity solid phase - Google Patents
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- CN102089662B CN102089662B CN200980127211.9A CN200980127211A CN102089662B CN 102089662 B CN102089662 B CN 102089662B CN 200980127211 A CN200980127211 A CN 200980127211A CN 102089662 B CN102089662 B CN 102089662B
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Abstract
A high capacity solid phase useful for bioaffinity assays and other solid phase applications prepared by coating a solid support with an analyte-specific biomolecule in the presence of a zwitterionic additive is displayed. Structures prepared according to the invention provide high capacity solid phases with enhanced binding properties, which are advantageous for any solid phase based assays. The present invention furthermore relates to the use of such solid phase and a method for the production thereof.
Description
The present invention relates to for the high capacity solid phase that bioaffinity measures and other solid phase is applied.Purposes that the invention still further relates to this high capacity solid phase and preparation method thereof.
Solid phase biological compatibility measures according to by antibody or other Biomolecular adsorption on a solid support, such as at the outside surface of spheric grain, or on the inside surface of test tube, microtiter well or plane " chip " surface, then it catch molecule or analysis thing to be measured for fixing second.
Now, known many different coating techniques functional activity surface of applying for the preparation of solid phase.But, current to measuring sensitivity, the expectation of dynamics and durable (robustness) proposes new demand key being measured to composition, such as solid phase character.Existing model surface usually can not meet the requirement for measuring performance and Geng Gao surface conjunction capacity.
The most straightforward procedure producing solid-phase coating is the protein adsorbing unmodified on a solid support, and described solid carrier is usually made up of polystyrene, polypropylene or some other plastic materials or glass.By proteolytic in damping fluid, such as carbonate or phosphate buffer, be then applied to the surface of solid carrier.Described be coated on moistening environment under carry out, then utilize saturation process block nonspecific binding site, final drying surface.But owing to usually only there being fraction native protein to retain its activity after this passive adsorption, therefore in binding capacity, the solid-phase coating obtained in this approach is not usually optimal selection.
Therefore, adsorption property and the surface stability of improving protein bag quilt are sought in multinomial research, and such as protein is coupled to different " carrier " protein before being adsorbed on solid carrier.Usually need to modify macromolecular natural structure to produce the adsorption property improved, meet the demand recorded.
This type of large molecule a kind of is streptavidin, and it is widely used in and catches biotinylated protein in solid phase.Comprise during microarray, biology sensor and other bioaffinity measure in many application based on solid phase and applied streptavidin and to combine closely the fundamental property of biotin.
US 5,061,640A describes the purposes of the adsorbability strengthening streptavidin compared with larger precursor protein.Described method provides method for coating, it is by being connected to more hydrophobic carrier molecule as on bovine serum albumin(BSA) by streptavidin, then be coated on hydrophobic solid carrier and improve streptavidin attachment on a solid support, therefore obtain that there is the solid phase improving binding property.
The other method improving the adsorption property of coated material is described in US 6,638, in 728B1, which depict being polymerized of monomer streptavidin and bi-functional cross-linking agent, to provide the dimer of streptavidin, tripolymer and tetrameric potpourri, when being adsorbed on polystyrene, its biotin binding capacity with improvement more surperficial than natural streptavidin.
(thiolated) streptavidin of sulfhydrylation has also shown through the polymerization of disulfide group (disulphide groups) improvement providing solid phase character, when being coated on polystyrene surface, by minimize between incubation period protein from solid phase spill and produce enhancing surface stability (
l., Laurikainen, K., 2006: " Comparison-study of streptavidin coated microtitrationplates ", J.Immunol.Methods.308,203).But this research also discloses, be better than the solid phase character of natural streptavidin and the binding capacity of improvement although be shown based on the bag of the streptavidin of sulfhydrylation, but other bag is shown higher adsorption capacity.On the other hand, described bag is tending towards unstable.
In fact, solid phase character height depends on bag used by condition, the buffer composition that described condition comprises application and the coating technique used.Therefore, multinomial research has disclosed the importance that Buffer additive in printing solutions improves some form (spot morphology) and repeatability.In order to reduce difference between a form and improve the overall repeatability of some product (spot production), usually use different surfactants.
Therefore, for US 2006/0223074A1, proposed for wrapping by the spotting solution of solid carrier (spotting solution), it comprises aklylene glycol, betaine, washing agent and salt.Betaine has zwitter-ion structure, and washing agent is ion-type or nonionic disclosed in US 2006/0223074.The composition provide some consistance (spot consistency) and the stability of height, and be convenient to produce and long-term preservation.
Other research reports the buffer composition of the multinomial optimization about DNA-microarray.But, although when these researchs report the adjuvant when using betaine as spotting solution, make moderate progress than the conventional spotting solution signal intensity not containing adjuvant, this type of is packaged as less total binding capacity.
Therefore still exist for the requirement that solid carrier provides solid-phase coating, wherein said solid phase shows that high binding capacity does not but reduce Properties in Stability.
Therefore an object of the present invention is to provide this type of high capacity solid phase, it can be used as efficient application such as microarray, biology sensor and other bioaffinity and measures.
Therefore first aspect of the present invention relates to high capacity solid phase, and it utilizes the method preparation comprising next coming in order step:
A) under the existence of zwitter-ion adjuvant, with analysis thing-specific biological molecules bag by solid carrier, wherein said biomolecule comprises SH-group,
B) solid carrier grace time is hatched, so that described biomolecule is fixed to described solid carrier, and
C) dry described solid carrier.
Within a context, term " high capacity solid phase " used herein refers to the suitable solid phase of any applicable biologicall test.
In addition, within a context, term " solid carrier " used herein refers to any being applicable to by the Solid carriers suitable of above-mentioned high capacity solid phase bag quilt.Typically, this type of solid carrier can be polystyrene, polypropylene or some other plastic material or glass.
Term used herein " is analyzed thing-specific biological molecules " and is referred to any biomolecule analysis thing to specific binding capacity.Should be understood that described biomolecule is special by directly combining analysis thing, such as, use antibody to be bonded to analysis thing specifically as biomolecule.Or described biomolecule can be bonded to analysis thing indirectly, such as usage chain Avidin combines the biotin moiety be connected in test analyte as biomolecule.
Should be understood that the SH-group that biomolecule comprises can be present in the natural structure of biomolecule used according to the present invention, such as, with the form of the disulfide bond reduced in biomolecule, or it produces in natural protein structure by chemical modification.
Term " zwitter-ion adjuvant " used herein refers to any electroneutral polar compound but carrying positive and negative charge on different atoms.The embodiment of zwitter-ion adjuvant is betaine and Phosphorylcholine.
For high capacity solid phase of the present invention, provide the solid phase with surprising enhancing binding property.Therefore, shockingly observe, use and be buffered liquid (namely comprising zwitter-ion adjuvant and the biomolecule containing SH-group) bag by solid phase according to the bag of novelty of the present invention, when using this bag by solution with dry coating technique (dry coatingtechnique), be provided than the bag of routine the binding activities highly increased.Therefore this bag is shockingly provided the highly reinforcing adsorption property of coated material (improving up to 100 times), and therefore makes it possible to prepare the high density solid phase with high power capacity binding property.
In addition, the some form of improvement is considered as the high stability of the solid phase obtained.
Utilize following methods to obtain according to solid phase of the present invention, described being coated on is hatched middle drying, namely before saturation process for the first time.This coating technique is " dry bag quilt ".The quick method for coating that dry bag is caught easily to carry out becomes possibility, and is operated compared with the bag in routine the T.T. shortened needed for method for coating.Dry bag can be applied with the some method for coating of routine (such as distributing and the printing of solid pin) and other printing process (such as ink jet printing) conversely.
In certain embodiments of the present invention, the step preparation comprising the nonspecific binding site used on the saturated solid carrier of non-interaction composition is further utilized according to high capacity solid phase of the present invention.
Term " non-interaction composition " used herein refers to not with biomolecule used according to the invention or analyzes the interactional organic compound of thing generation specificity or the second biomolecule, and namely described non-interaction composition is only combined on nonspecific binding site.Typically, this non-interaction composition is inert protein, such as bovine serum albumin(BSA), and it will be combined on the nonspecific binding site of solid carrier.And non-interaction composition can be combined on the nonspecific proteins matter site of solid phase.Therefore, the signal to noise ratio (S/N ratio) of specificity and non-specific binding is improved, and obtained detection system is sensitiveer to detected analysis thing.
Analysis thing-specific biological molecules according to the present invention can be selected from protein, antibody, antibody fragment, antigen, receptors ligand, protein acceptor, binding proteins specific matter, fit, nucleic acid, oligonucleotides and peptide.
Within a context, term " antibody " used herein refers to any immunoglobulin (Ig) or its fragment, and comprises any polypeptide containing antigen binding site.Described term include but not limited to polyclone, monoclonal, monospecific, polyspecific, non-specific, humanized, people, strand, chimeric, synthesis, restructuring, hydridization, sudden change, transplant with the antibody of external generation.
For different mensuration, to organize in biomolecule arbitrary relevant advantage known to those skilled in the art to this.
Streptavidin is a kind of conventional biomolecule.
In some embodiments, described biomolecule is not nucleic acid.
In certain embodiments of the present invention, described biomolecule before bag quilt through Thiolation modification.
Should be understood that functional SH-group in biomolecule can be produced by the endogenous disulfide bridge in reduction biomolecule, such as, by enzyme or chemical cracking antibody or other oroteins, to discharge the antibody fragment containing reactive sulfydryl (sulphydryl).Or the suitable thiolating reagent generating new sulfydryl in biomolecule is undertaken by any by thiolation.
In one embodiment, described biomolecule contains the SH-group in Already in biomolecule.
In one embodiment, described biomolecule is modified by the endogenous disulfide bridge in reduction biomolecule and introduces SH-group.
In one embodiment, described biomolecule is modified by the sulfenyl reduced in Already in biomolecule and introduces SH-group.
In certain embodiments of the present invention, described biomolecule is modified by the thiolation of any suitable thiolating reagent and introduces SH-group, such as thiolating reagent is selected from S-acetyl thioglycolic acid (S-acetylthioacetic acid, SATA), 3-(2-pyridyidithio) propionic acid N-hydroxy-succinamide ester (SPDP), N-succinimidyl-S-acetyl mercaptopropionic acid ester (N-succinimidyl-S-acetylthiopropionate, SADP), succinimidyloxycarbonyl-Alpha-Methyl-α-(2-pyridyidithio) toluene (SMPT) and 2-imido grpup thiacyclopentane (Traut reagent).
Use this thiolating reagent, the effective and stable Thiolation of biomolecule can be obtained.
In certain embodiments of the present invention, described zwitter-ion adjuvant has following basic structure:
A’-R-A”
Wherein,
A ' is selected from
Wherein R
1, R
2and R
3be hydrogen, halogen ,-NO independently
2,-CN ,-OR
4,-SR
4,-SO
2r
4,-SOR
4, C
1-6-alkyl, C
1-6-Polyfluoroalkyl, C
2-6-thiazolinyl, C
2-6-alkynyl, C
3-6-naphthenic base, C
2-6-alkoxyalkyl, C
2-6-alkyl alkylthio base (alkylthioalkyl), C
2-6-alkylaminoalkyl group, wherein R
4for hydrogen or C
1-6-alkyl,
R is formula-(CH
2)
m-alkyl, and m=0-22, and
A " be selected from-O
-,-CO
2 -,-OSO
3 -,-OPO
3 2-with-OPO
3h
-.
In a concrete embodiment, R
1, R
2and R
3be hydrogen independently, or identical or different formula CH
3-(CH
2)
nthe alkyl of-expression, and n=0-5.
In concrete embodiments more of the present invention, R
1, R
2and R
3be not hydrogen.
Therefore, in these embodiments of the present invention, described zwitter-ion adjuvant has the structure similar to betaine, and it provides effective and stable bag quilt.
In some embodiments, described zwitter-ion adjuvant is selected from betaine and Phosphorylcholine.Term " betaine " used herein refers to concrete compound N, N, Betaine.
In certain embodiments of the present invention, described bag is utilized and is selected from following method for coating and carries out: point distributes bag by, solid pin printing and ink jet printing.
When the concept according to dry bag quilt is applied, this coating technique allows high reappearance bag quilt, to prepare high capacity solid phase of the present invention.
Method for coating also can comprise passive adsorption, through function base or biochemical interactional chemical coupling.
Another aspect of the present invention relates to high capacity solid phase according to the present invention and is detecting the purposes in the method for the analysis thing in sample for solid phase, said method comprising the steps of:
A) sample of analysis thing content to be detected is added high capacity solid phase prepared in accordance with the present invention, and
B) existence of analyte or its concentration.
Measure or detection specificity be bonded to the method for the analysis thing of high capacity solid phase known to those skilled in the art.It completes by passive method, is bonded to such as detection specificity the content of the analysis thing of solid phase, such as, use UV spectrophotometric method by the content of UV absorptiometry protein, peptide, amino acid and/or nucleic acid.Or and preferably, detect or analyte by using the label be present in detection system.Described label can be present in analyze on thing, the gametophyte (partner) of biomolecule or be bonded to analyze thing the second biomolecule on.
Described label can be the endogenous enzyme part (member) analyzing thing self, or there is the composition (moiety) of enzymatic activity or radioactively labelled substance or other suitable detectable signal response component any, its by light splitting, optics, mode that is photochemical, immunochemical, electric, heat or chemistry detects.
In some embodiments, fluorescent dye is labeled as described in.In some embodiments, described fluorescent dye is selected from Rare Earth Chelate as Europium chelate; Fluorescein type comprises FITC, CF, 6-Fluoresceincarboxylic acid; Rhodamine type comprises TAMRA, dansyl, Liz amine, cyanine, phycoerythrin; Texas Red and analog thereof.
In some embodiments, described label is radioactive nuclide, and it is selected from
3h,
11c,
14c,
18f,
32p,
35s,
64cu,
68ga,
86y,
99tc,
111in,
123i,
124i,
125i,
131i,
133xe,
177lu,
211at,
213bi and
51cr.
According to certain embodiments of the present invention, described mark be present in analyze on thing, on the gametophyte of the biomolecule of solid phase or be bonded to analyze thing the second biomolecule on.
With relevant advantage arbitrary in these embodiments known to those skilled in the art.
In certain embodiments of the present invention, by linking parsing thing to the binding partners of biomolecule, described analysis thing is made to have specificity to biomolecule.
Therefore, in the preferred embodiment of the present invention, described binding partners is biotin and described biomolecule is selected from Avidin and streptavidin.Or described binding partners is selected from Avidin and streptavidin and described biomolecule is biotin.
In certain embodiments of the present invention, described biomolecule is antibody or the antibody fragment of antigentic specificity, and described antigen is binding partners.
In certain embodiments of the present invention, described analysis thing is directly also bonded to described biomolecule specifically.In some embodiments, described biomolecule is antibody or antibody fragment, and it is bonded to analysis thing specifically.
With relevant advantage arbitrary in these embodiments known to those skilled in the art.Therefore, each embodiment of the present invention provides the height effective use of high capacity solid phase.
According to a third aspect of the invention we, provide the method preparing high capacity solid phase, wherein said method comprises next coming in order step:
A) under the existence of zwitter-ion adjuvant, with analysis thing-specific biological molecules bag by solid carrier, wherein said biomolecule comprises SH-group,
B) solid carrier grace time is hatched, so that described biomolecule is fixed to described solid carrier, and
C) dry described solid carrier.
The present invention is open in more detail by reference to accompanying drawing, wherein,
Fig. 1 illustrates the biotin binding capacity of the spot face (spot surfaces) of streptavidin bag quilt, uses the biotin of europium mark and large molecule that is biotinylated and europium mark respectively.
Fig. 2 illustrates the biotin binding capacity of the microtiter wells of the C-form (C-format) of streptavidin bag quilt, described microtiter wells uses the streptavidin preparation of unmodified and contains in bag is by solution or do not contain adjuvant, or uses the streptavidin preparation of sulfhydrylation and contain adjuvant in bag is by solution.
Fig. 3 illustrates the biotin binding capacity of the loading wells (spot wells) of the streptavidin bag quilt of sulfhydrylation, and described loading wells uses point distribution method preparation, and it is compared with the streptavidin of unmodified.
Fig. 4 shows protein thiolation and betaine as bag by the adjuvant in solution, to provide the purposes of the improvement surface nature of the antibody bag quilt on polystyrene, described antibody bag is used the antibody of natural antibody or sulfhydrylation, obtains after the tracer antibody adding cTnI and europium mark.
Fig. 5 illustrates the fragment antibody of sulfhydrylation and the betaine bag at antibody bag quilt by the purposes in solution, and described antibody bag is used contact print technology to prepare on polystyrene.
The specific signals of fluorescent light source that Fig. 6 display measures from oligonucleotides, described in be determined at high power capacity spot face and carry out on the surface at the contrast point sample using contact print technology to prepare.
Following non-limiting example is exemplified with the present invention.
Embodiment 1
The preparation of the streptavidin of sulfhydrylation and utilize contact print bag quilt
Streptavidin (4mg) is dissolved in phosphate buffer (50mM NaH
2pO
4/ Na
2hPO
4, pH7.5) and to the final concentrations of 148 μMs.The N-hydroxy-succinamide ester of dual-functional group crosslinking aid S-acetyl thioglycolic acid (SATA) is dissolved in dimethyl formamide, then adds in reaction, to provide SATA more excessive than streptavidin 30 times moles.To react incubated at room 30 minutes.After hatching, 50mM azanol is used to mix the sulfydryl deprotection 2 hours of the protection of protein amine.Then through the end-product of desalting column (desalting column) purifying containing the streptavidin of sulfhydrylation, and with containing 50mM borate buffer solution (pH8.3) wash-out of 1mM EDTA.The existence of the sulfydryl using Ellman ' s reaction checking not paired, and general discovery per molecule streptavidin on average mixes 10-12 sulfydryl.
The streptavidin of above-mentioned sulfhydrylation is buffered liquid (100mM Na at bag
2hPO
4/ 50mM citric acid, 0.5M betaine) in be diluted to the concentration of 1.5mg/ml, and for the preparation of the high power capacity bag quilt on polystyrene microtiter wells.Described solution uses solid pin printing technology to utilize 2.0mm solid pin to apply.After printing, at+35 DEG C, the hole of printing points is hatched and dried overnight.After overnight incubation, use cleaning solution (5mM Tris-HCl (pH7.75), 9g/l NaCl, 1g/l Germall II and 0.05g/l polysorbas20) washing hole, and use containing 50mM Tris-HCl (pH7.0), 150mM NaCl, 0.5g/lNaN in room temperature
3, 60g/l D-D-sorbite and 2g/l bovine serum albumin(BSA) the saturated 2-20 hour of blocking solution.After saturated, hole is for subsequent use in dry 5 hours of fuming cupboard.
Use the biotin binding capacity (i.e. fixed efficiency) of two reporter assay loading wells: the biotin (Bio-Eu) of europium-mark and carry the monoclonal antibody (Bio-Mab-Eu) of biotin and europium-mark.Undersized Bio-Eu makes to be bonded to sterically hindered site, and therefore it simulates total biotin binding capacity.At mensuration damping fluid, (Innotrac buffer soln is red, from Innotrac Diagnostics, Turku, Finland) in, the dilution series of preparation Bio-Eu (0.2nM to 10nM) and Bio-Mab-Eu (0.1nM to 60nM), is then added to each hole with the volume in 50 μ l/ holes.By hole under shake in incubated at room 1 hour, is then used the 5mMTris-HCl (pH 7.75) containing 9g/l NaCl, 1g/l Germall II and 0.005g/l polysorbas20 to wash six times.In hot blast (60 DEG C) after dry 5 minutes, use Victor multiple labeling counter (multilabel counter) (PerkinElmer Life Sciences-Wallac Oy, Turku, Finland) from the bottom in the hole of drying, direct Measuring Time differentiates (time-resolved) fluorescence intensity.
Fig. 1 shows the fluorescence signal of the counting per second (cps) of the concentration for Bio-Eu (" Bio-Eu " in figure) and Bio-Mab-Eu (" Bio-Ab-Eu " in figure) respectively.For both Bio-Eu and Bio-Mab-Eu, (sulfhydrylation with betaine process) (being respectively B and D) streptavidin of above-mentioned modification is compared with natural streptavidin (being respectively A and C).Therefore, compare with the spot face using conventional chemical to prepare (namely not added the streptavidin of betaine in solution without the streptavidin of sulfydryl derivatization and bag), the binding capacity of the loading wells described in embodiment 1 for Bio-Eu up to 100 times and for about 40 times of Bio-Mab-Eu large molecule height.Use another kind of surfactant Phosphorylcholine, the binding capacity on streptavidin surface is increased to 100 times for Bio-Eu and Bio-Mab-Eu is increased to 30 times of (not shown)s.
Embodiment 2
For improving the binding capacity of the microtiter wells of the conventional C-form of streptavidin bag quilt, betaine and protein thiolation are wrapping by the purposes in solution
This embodiment illustrates betaine is added agent purposes as the bag for wrapping quilt, described bag is used the bag of 200 μ l to be prepared in the microtiter wells of conventional C-form by volume.Described experiment compare use unmodified prepare with the streptavidin of sulfhydrylation, with or the binding capacity of streptavidin plate of bag quilt of non-garden beet alkali.
Streptavidin is buffered liquid (100mM Na at bag
2hPO
4/ 50mM citric acid, containing or not containing extra 0.5M betaine) in be diluted to the final concentration of 5 μ g/ml, and add microtiter wells (Maxisorp microtiter wells, NUNC A/S, Denmark) with the volume in 200 μ l/ holes.At+35 DEG C by this hole overnight incubation.Dry in process bag by solution is hatched what spend the night.Use next day cleaning solution to wash this hole, and spend the night room temperature is saturated as described in Example 1.Absorb saturated solution and by hole at dry several hours (2-5h) of fuming cupboard.
Use the biotin binding capacity in this hole of determination of biotin of europium mark.Bio-Eu is being measured in damping fluid (Innotrac buffer soln is red) concentration being diluted to 0.1 and 1 μM, and is adding in the hole of streptavidin bag quilt with the volume in 200 μ l/ holes.Experimental program is as described in example 1 above used to carry out in this test of other side.
Fig. 2 shows the binding capacity (by fluorescence measurement, in cps) of A (natural streptavidin), B (natural streptavidin and betaine) and C (streptavidin of sulfhydrylation and betaine).The streptavidin plate being coated with extra betaine provides the improvement of 3 times in biotin binding capacity.When using the streptavidin of sulfhydrylation and use betaine in bag is by solution, obtain the improvement of 14 times of binding capacity.
Embodiment 3
Wrap by betaine and protein thiolation improving the purposes of binding capacity of loading wells of the streptavidin bag quilt utilizing point distribution method to prepare
This embodiment illustrates and print the purposes of different spotting methods in the loading wells preparing the binding capacity with enhancing from solid pin.Described loading wells instills 4 μ l bags by solution preparation (i.e. point distribution method) in the middle of bottom microtiter wells.Contrasted using the streptavidin of sulfhydrylation and the bag containing adjuvant with the binding capacity contrasting spot face of the streptavidin of unmodified by the binding capacity of high power capacity spot face prepared by solution.
Use experimental program as described in Example 1 to modify streptavidin to make it containing sulfydryl.The streptavidin of sulfhydrylation is dissolved in bag and is buffered final concentration to 1.5mg/ml in liquid (containing 0.5M betaine).By use conventional pipette (Finnpipette), the streptavidin solution of 4 μ l sulfhydrylations is applied to the bottom of microtiter wells.The bag used is identical with described in embodiment 1 by condition.Use experimental program as described in Example 1, use Bio-Eu to measure the biotin binding capacity in the hole of bag quilt.
Fig. 3 shows the binding capacity (by fluorescence measurement, in cps) of A (natural streptavidin) and B (streptavidin of sulfhydrylation and betaine).With use compared with the loading wells prepared of natural streptavidin, wrap by extra betaine, the streptavidin of sulfydryl modification illustrates at least 27 times of improvement in biotin binding capacity.
Embodiment 4
Antibody bag on the spot face that betaine and protein thiolation are prepared in contact print by purposes
The present embodiment illustrate betaine as bag be added agent the antibody bag using contact print technology to carry out by purposes.The fixed efficiency that illustrated mensuration compares the monoclonal anti surface that the high power capacity SH-that uses extra betaine to prepare modifies with need not the fixed efficiency of contrast surface prepared of extra betaine.
SATA reagent is used to modify monoclonal antibody (anti-cTnI monoclonal antibody), to mix extra sulfydryl in protein.2mg monoclonal antibody is dissolved in phosphate buffer (50mM NaH
2pO
4/ Na
2hPO
4; PH7.5) to the final concentration of 28 μMs.SATA is dissolved in dimethyl formamide and adds in described reaction, SATA is provided more excessive than monoclonal antibody 50 times moles.After within 30 minutes, hatching, 50mM azanol is used to mix the protection sulfydryl deprotection that formed in antibody 2 hours.Then by desalting column purification reaction potpourri, and by the monoclonal antibody of product, sulfhydrylation at the middle wash-out of 0.1M sodium phosphate buffer (pH7.2) containing EDTA.
The antibody of sulfhydrylation is buffered in liquid at bag the concentration being diluted to 0.5mg/ml, then applies contact print and use the solid pin of 2.0mm diameter to be applied to the bottom of microtiter wells (Maxisorp, NUNC A/S, Denmark).The loading wells of further pack processing quilt as described in Example 1.Use cTnI-Immunoassays measure wrap by the performance of the immunoassays in hole.At 7.5%BSA-TSA damping fluid (50mMTris-HCl, pH7.75; 154mM NaCl; 0.05g/l NaN3; 75g/l BSA) in prepare the dilution series of cTnI antigen with the concentration of 0.001 to 800ng/ml.20 μ l tracer agent antibody (the anti-cTnI monoclonal antibody of insolubilized antibody and mark europium is combined in the different epi-positions of cTnI) and 20 μ l standard dilution are added bag quilt loading wells and under shaking (900rpm) hatch 1 hour in+36 DEG C.After hatching 1 hour, use cleaning solution that hole is washed 6 times, and under hot blast (+60 DEG C) dry 5 minutes, then measuring-signal.Use Victor multiple labeling counter directly from Measuring Time resolved fluorometric signal bottom the hole of drying.
Fig. 4 shows the cTnI immunoassays signal (measuring fluorescence, in cps) of A (normal antibody) and B (sulfydryl modification with the antibody of betaine process).In cTnI immunoassays, in contrast to contrast surface, use the antibody of sulfhydrylation and the hole of extra betaine bag quilt to provide in specific signals level (subtracting background signal) improvement up to 66%.
In addition, not used compared with the surface of native gene antibody bag quilt by solution containing the bag of betaine with using, use according to the fragment antibody of the sulfhydrylation of above-mentioned similar operations and use betaine in bag is by solution, the surface conjunction capacity of cTnI provides the improvement of 50 times to 60 times.As shown in Figure 5, which show the cTnI measured signal (in cps) of A (normal fragment antibody) and B (sulfhydrylation with the fragment antibody of betaine process).
Embodiment 5
High power capacity streptavidin bag is being caught the purposes in biotinylated oligonucleotides by (streptavidin of sulfhydrylation and betaine)
Use the some bag of the streptavidin of the sulfhydrylation of method of contact printing as described in Example 1 preparation containing betaine adjuvant surperficial.For contrast surface, employ and do not wrap by hole containing the streptavidin preparation point of the sulfhydrylation of betaine adjuvant.Method of contact printing is used to prepare control wells in the mode (not using betaine adjuvant) identical with high power capacity surface.
By biotinylated oligonucleotides measure in damping fluid (the 1M NaCl containing extra) be diluted to 0.0002,0.0001,0.002,0.01,0.02, the concentration of 0.04pmol/ μ l, and to be applied on spot face with the volume of 50 μ l/ loading wells.At room temperature hatching this hole 30 minutes with slowly shaking, hatching rear use cleaning solution (5mM Tris-HCl (pH7.75), 9g/l NaCl, 1g/l Germall II and 0.05g/l polysorbas20) and hole is washed twice.The second oligonucleotides marked by terbium (Tb) dilutes the concentration of 0.03pmol/ μ l in damping fluid measuring, and be applied to this loading wells.With shake (900rpm), 1.5h is hatched in this hole at 35 DEG C, hatch rear use cleaning solution and this hole is washed 4 times.After the Delfia enhancing of 20 minutes, service time, resolved fluorometer measurement derived from the signal of terbium.
Fig. 6 shows the measured signal (measurement fluorescence, in cps) of A (oligonucleotides (oligonucleotid) that the streptavidin of sulfhydrylation catches) and B (oligonucleotides caught with the streptavidin of betaine process of sulfydryl modification).In addition, Fig. 6 shows the sample room difference (i.e. CV% value, n=3) of A (C) and B (D).
Be compared to and use the streptavidin (steptavidin) of sulfhydrylation and do not add spot face prepared by betaine, use the streptavidin of sulfhydrylation and add the remarkable improvement that spot face prepared by betaine provides the specific signals obtained in oligonucleotides measures.With contrast wrap by compared with, use high power capacity is coated on the special signal aspect deriving from terbium and obtains improvement up to 5 times.Except the improvement of specific signals level, use high power capacity point bag has been reduced significantly the difference between the same form many parts of holes.
Claims (15)
1. utilize high capacity solid phase prepared by the method for coating comprising next coming in order step:
A) applied on a solid support by bag by solution, wherein said bag is included in the analysis thing-specific biological molecules under the existence of zwitter-ion adjuvant by solution, wherein said biomolecule comprises SH-group,
B) to hatch and dry described solid carrier grace time, so that described biomolecule is fixed to described solid carrier, and
C) with the nonspecific binding site on the saturated solid carrier of non-interaction composition,
Wherein said analysis thing-specific biological molecules is selected from protein, antigen, receptors ligand, protein acceptor, fit, nucleic acid and peptide.
2. solid phase according to claim 1, wherein said analysis thing-specific biological molecules is selected from antibody, antibody fragment, binding proteins specific matter and oligonucleotides.
3. solid phase according to claim 1, wherein said analysis thing-specific biological molecules is streptavidin.
4. solid phase as claimed in one of claims 1-3; wherein said biomolecule has been modified through thiolation and has been introduced SH-group, and thiolating reagent used is selected from S-acetyl thioglycolic acid (SATA), 3-(2-pyridyidithio) propionic acid N-hydroxy-succinamide ester (SPDP), N-succinimidyl-S-acetyl mercaptopropionic acid ester (SADP), succinimidyloxycarbonyl-Alpha-Methyl-α-(2-pyridyidithio) toluene (SMPT) and 2-imido grpup thiacyclopentane (Traut reagent).
5. solid phase according to claim 1, wherein said zwitter-ion adjuvant has following basic structure:
A’-R-A”
Wherein,
A ' is selected from
Wherein R
1, R
2and R
3be hydrogen, halogen ,-NO independently
2,-CN ,-OR
4,-SR
4,-SO
2r
4,-SOR
4, C
1-6-alkyl, C
1-6-Polyfluoroalkyl, C
2-6-thiazolinyl, C
2-6-alkynyl, C
3-6-naphthenic base, C
2-6-alkoxyalkyl, C
2-6-alkyl alkylthio base, C
2-6-alkylaminoalkyl group, wherein R
4for hydrogen or C
1-6-alkyl,
R is formula-(CH
2)
m-alkyl chain, and m=0-22, and
A " be selected from-O
-,-CO
2 -,-OSO
3 -,-OPO
3 2-with-OPO
3h
-.
6. solid phase according to claim 5, wherein R
1, R
2and R
3be hydrogen independently, or identical or different formula CH
3-(CH
2)
nthe alkyl group of-expression, and n=0-5.
7. solid phase as claimed in one of claims 1-3, wherein said applying step is undertaken by following application process: point distributes bag quilt, the printing of solid pin and ink jet printing.
8. solid phase according to claim 4, wherein said applying step is undertaken by following application process: point distributes bag quilt, the printing of solid pin and ink jet printing.
9. according to the solid phase of claim 5 or 6, wherein said applying step is undertaken by following application process: point distributes bag quilt, the printing of solid pin and ink jet printing.
10. high capacity solid phase as claimed in one of claims 1-9 is detecting the purposes in the method for the analysis thing in sample for solid phase, said method comprising the steps of:
A) sample of analysis thing content to be detected is added high capacity solid phase as claimed in one of claims 1-9, and
B) existence of analyte or its concentration.
11. purposes according to claim 10, wherein by detect be present in analyze on thing, on the gametophyte of biomolecule or the label be bonded in the second biomolecule analyzing thing measure described analysis thing.
12. purposes any one of claim 10 and 11, wherein said analysis thing is connected to the binding partners of described biomolecule.
13. purposes according to claim 12, wherein binding partners is biotin and described biomolecule is selected from Avidin and streptavidin.
14. purposes according to claim 12, wherein said biomolecule is antibody or the antibody fragment of antigentic specificity, and described antigen is binding partners.
15. method for coating preparing high capacity solid phase, wherein said method for coating comprises next coming in order step:
A) applied on a solid support by bag by solution, wherein said bag is included in the analysis thing-specific biological molecules under the existence of zwitter-ion adjuvant by solution, wherein said biomolecule comprises SH-group,
B) also drying solid carrier grace time is hatched, so that described biomolecule is fixed to described solid carrier, and
C) with the nonspecific binding site on the saturated solid carrier of non-interaction composition.
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| DKPA200801003 | 2008-07-16 | ||
| DKPA2008/01003 | 2008-07-16 | ||
| PCT/DK2009/000168 WO2010006604A1 (en) | 2008-07-16 | 2009-07-10 | High capacity solid phase |
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| US (1) | US8669120B2 (en) |
| EP (1) | EP2318838B1 (en) |
| JP (1) | JP5476376B2 (en) |
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| KR20120128440A (en) * | 2011-05-17 | 2012-11-27 | 삼성전자주식회사 | Kit and method for detecting target material |
| GB2516808A (en) * | 2013-05-31 | 2015-02-11 | Innova Biosciences Ltd | Antibody composition and buffer system therefor |
| CN111902720A (en) | 2018-03-21 | 2020-11-06 | 沃特世科技公司 | Non-antibody high affinity based sample preparation, adsorbents, devices and methods |
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| DE3640412A1 (en) | 1986-11-26 | 1988-06-09 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A SPECIFICALLY BINDABLE SUBSTANCE |
| WO1990008320A1 (en) | 1989-01-19 | 1990-07-26 | Teijin Limited | Immunoassay method, reagent and kit |
| JPH10253632A (en) * | 1997-03-10 | 1998-09-25 | Nissui Pharm Co Ltd | Method, kit and device for analysis |
| AU746325B2 (en) * | 1997-06-25 | 2002-04-18 | Innogenetics N.V. | Methods for covalent immobilisation of biomolecules to a carrier by means of a His-tag |
| PT1071955E (en) * | 1998-04-17 | 2005-02-28 | Innogenetics Nv | IMMUNOLOGICAL DIAGNOSTIC IMPROVED ASSAYS USING REDUCING AGENTS |
| AU5080699A (en) * | 1998-07-21 | 2000-02-14 | Burstein Laboratories, Inc. | Optical disc-based assay devices and methods |
| US6638728B1 (en) | 2000-06-16 | 2003-10-28 | Pierce Biotechnology, Inc. | Coated surfaces with high capacity for capturing target molecules |
| US20030149246A1 (en) * | 2002-02-01 | 2003-08-07 | Russell John C. | Macromolecular conjugates and processes for preparing the same |
| WO2004046687A2 (en) * | 2002-11-14 | 2004-06-03 | Qtl Biosystems, Llc | Methods of biosensing using fluorescent polymers and quencher-tether-ligand bioconjugates |
| CN102841195B (en) * | 2005-12-21 | 2016-02-03 | 梅索斯卡莱科技公司 | There is analysis module and the preparation and application thereof of analytical reagent |
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| ELISA抗原的"干包被"方法试验;陈兴扶 等;《中国动物检疫》;19901231(第6期);摘要，第12页第5段 * |
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| US8669120B2 (en) | 2014-03-11 |
| EP2318838B1 (en) | 2013-09-04 |
| DK2318838T3 (en) | 2013-10-14 |
| US20100173425A1 (en) | 2010-07-08 |
| JP2011528106A (en) | 2011-11-10 |
| CN102089662A (en) | 2011-06-08 |
| JP5476376B2 (en) | 2014-04-23 |
| WO2010006604A1 (en) | 2010-01-21 |
| EP2318838A1 (en) | 2011-05-11 |
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